CRISPRi lentiviral sgRNAワークフロー

CRISPRi dCas9-SALL1-SDS3 lentiviral particlesを使用して安定したdCas9-SALL1-SDS3発現細胞株を確立し、CRISPRi lentiviral sgRNA particles（左）またはplasmid sgRNA（右）を導入するワークフローです。

CRISPRiコントロール

CRISPRi lentiviral sgRNAポジティブコントロール

• 十分に特徴付けられた遺伝子を標的とするCRISPRi sgRNAを用いて、最大の発現抑制を得るための実験条件の検討を行います。

CRISPRi lentiviral sgRNA non-targetingコントロール

• ターゲット特異的sgRNAの非存在下でCRISPRiコンポーネントに対するベースライン細胞応答を評価するためのCRISPRi non-targetingコントロールです。

関連試薬

CRISPRi dCas9-SALL1-SDS3

• ヌクレアーゼ不活性化Cas9を発現するレンチウイルス粒子またはmRNAは、当社独自の転写抑制因子に融合しています。

CRISPRiレンチウイルスsgRNAは長時間転写抑制に最適である

dCas9-KRABまたはdCas9-SALL1-SDS3を安定して発現するU2OSおよびA549細胞を、24ウェルプレートに50,000細胞/ウェルでプレーティングし、PPIBあるいはSEL1LをターゲットとするCRISPRi sgRNA lentiviral particlesをMOI 0.3で形質導入して、単一のインテグラントを含む細胞を得ました。細胞を2µg/mLピューロマイシンで5日間選択培養し、2つの集団に分割し、さらに1日回復培養しました。形質導入の7日後に、RT-qPCR分析のために1つの細胞集団を回収しました。もう１つの複製集団は、回収前にさらに7日間培養しました。回収したプレートから全RNAを単離し、ハウスキーピング遺伝子としてGAPDHを用いたΔΔCq法で相対的な遺伝子発現を計算し、non-targetingコントロールに対して正規化しました。

1. M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife. 5, e19760 (2016).

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Supporting Data

Improved CD8+ T-cell SMARTvector™ shRNA lentiviral system transduction using LentiBOOST™ Lentivirus Transduction Enhancer

100,000 primary human CD8+ T cells were transduced with either 30,000 (MOI 3, green) or 70,000 (MOI 7, purple) TUs of SMARTVector™ mCMV tGFP Lentiviral Control Particles targeting either NTC or PPIB along with 1:100 LentiBOOST transduction enhancer. Cells were centrifuged at 800 x g for one hour at 32 °C followed by a four hour incubation prior to removal of lentiviral particles and transduction enhancer. Transduction efficiency (%GFP+ out of live cells) and viability were determined 5 days post-transduction by flow cytometry. The addition of LentiBOOST technology markedly improved transduction efficiencies without significantly impacting cell viability.

Improved CD4+ and CD8+ T-cell Edit-R™ All-in-one sgRNA/Cas9 lentiviral system transduction using LentiBOOST™ Lentivirus Transduction Enhancer

100,000 primary human CD4+ and CD8+ T cells from two donors were transduced with 250,000 TUs of Edit-R GFP Delivery controls mCMV along with 1:100 LentiBOOST transduction enhancer. Cells were centrifuged at 800 x g for one hour at 32 °C followed by an overnight incubation prior to removal of lentiviral particles and transduction enhancer. Transduction efficiency and viability were determined 72 hours post-transduction by flow cytometry. The addition of LentiBOOST technology markedly improved transduction efficiencies without significantly impacting cell viability.

Improved transduction of human induced pluripotent stem cells (hiPSCs) with the Strict-R™ Inducible CRISPRa lentiviral system transduction using LentiBOOST™ Lentivirus Transduction Enhancer

10,000 WTC hiPS cells were transduced with either 20,000 (MOI 2, green) or 40,000 (MOI 4, purple) TUs of Strict-R™ Inducible EGFP dCas9-VPR Lentiviral Particles along with 1:100 LentiBOOST transduction enhancer. Cells were centrifuged at 800 x g for one hour at 32 °C followed by an overnight incubation prior to removal of lentiviral particles and transduction enhancer. Transduction efficiency and viability were determined 72 hours post-transduction by flow cytometry. The addition of LentiBOOST markedly improved transduction efficiencies without significantly impacting cell viability.